The groups of Miratul Muqit, Ian Ganley and Adrien Rousseau at the MRC-PPU in collaboration with Glenn Masson at the School of Medicine have identified novel crosstalk between the PINK1 mitophagy pathway and the integrated stress response pathway.
In previous work, the Muqit lab had identified phosphorylation of Rab8A at Serine 111 (pRab8A) as a downstream readout of PINK1 activation in cells and had developed a highly specific monoclonal antibody against pRab8A in partnership with the Michael J Fox Foundation and Abcam.
To investigate for kinase regulators of pRab8A, postdoc Ilaria Volpi, undertook a whole kinome siRNA screen in HeLa cells following mitochondrial depolarisation-induced PINK1 activation. Consistent with previous work, Ilaria observed that knockdown of PINK1 abolished endogenous pRab8A however, she found that knockdown of the integrated stress response (ISR) pathway kinase EIF2AK1 (also known as HRI) led to a significant increase in pRab8A and this was mediated by enhanced stabilisation of PINK1 associated with up-regulation of the PINK1 substrate phosphorylated ubiquitin (pUb). Taking these findings forward, postdocs Pawan Singh and Shalini Agarwal were able to show that HRI knockdown induced PINK1 stabilisation and activation in multiple different human cell lines including ARPE-19 and U2OS cells. Furthermore, they showed that this effect on PINK1 was specific to HRI and not seen with knockdown of other ISR kinases, EIF2AK 2 ,3 or 4.
Pawan and Shalini next investigated the mechanism and took advantage of discoveries by the Kampmann and Jae labs that mitochondrial damage induces cleavage of a mitochondrial protein DELE1 and the cleaved C-terminal fragment is released into the cytosol where it binds to and activates HRI leading to induction of the ISR and ATF4 transcriptional activation. Consistent with this they found that knockdown of DELE1 blocked ATF4 induction. Shalini was able to show that knockdown of DELE1 or HRI was associated with enhanced transcription and translation of PINK1. Furthermore, she found that the small molecule ISR inhibitor, ISRIB, also led to enhanced PINK1 levels and activation.
To determine the physiological consequence of the crosstalk between the ISR and PINK1 pathway, Lea Wilhelm, a postdoc in the Ganley lab, investigated the impact of genetic and chemical perturbation of the ISR on PINK1 mitophagy in ARPE-19 cells. Excitingly, Lea found that HRI knockdown or treatment with ISRIB enhanced PINK1-Parkin mitophagy and this was specific with no impact on another form of mitophagy induced by the iron chelator Deferiprone.
Overall, these studies suggest that HRI appears to act as a molecular brake in the PINK1 pathway and opens up completely unanticipated ways to develop new medicines that can reduce the accumulation of damaged mitochondria as a treatment for Parkinson’s and related brain disorders. Of clinical relevance, there are already drugs available like ISRIB which have proven to be safe in humans, and these findings indicate that these could be repurposed for testing in clinical trials of Parkinson’s patients right away.
The study was supported by funding from the Michael J Fox Foundation, Medical Research Council, EMBO and Wellcome Trust.
To read a copy of the paper published in Science Advances, click here.